The Era of Gene Therapy: The Advancement of Lentiviral Vectors and Their Pseudotyping.

Over 35 years of history, the field of gene therapy has undergone much progress. The initial concept-the replacement of dysfunctional genes with correct ones-has advanced to the next stage and reached the level of precise genome editing. Dozens of gene therapy products based on viral and non-viral delivery platforms have been approved, marking the dawn of the gene therapy era.

Establishment of human trophoblast stem cells from term smooth chorion.

Introduction: Human trophoblast stem (TS) cells derived from first-trimester placental villi (TS) and blastocyst (TS) are valuable for studying placental development and pathobiology. However, most placenta-mediated pregnancy complications are diagnosed in the third-trimester, and there are limited reports on TS cells from the third-trimester placental tissues. In this study, we report the successful derivation of TS cells from the term smooth chorion.

Structure and thickness of Glisson's capsule differ considerably on the liver surface in mammalian species.

Glisson's sheath is the connective tissue ensheathing the portal vein, hepatic artery, and bile duct within the liver. Although the connective tissue surrounding the liver surface is known as Glisson's capsule, its structure and function are poorly understood. In the present study, we analyzed mouse, rat, rabbit, feline, canine, monkey, porcine, bovine, and equine livers by histochemistry and immunohistochemistry analysis of α-smooth muscle actin, keratin 19, and podoplanin and examined how the structure of the Glisson's capsule is conserved and differs among the nine species.

The Dual-Pseudotyped Lentiviral Vector with VSV-G and Sendai Virus HN Enhances Infection Efficiency through the Synergistic Effect of the Envelope Proteins.

A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity.

Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells.

Similarities and differences in placental development between humans and cynomolgus monkeys.

Background: The placenta is an extraembryonic organ, which is essential to maintain a normal pregnancy. However, placental development in humans is poorly understood because of technical and ethical reasons.

Methods: We analyzed the anatomical localization of each trophoblastic subtype in the cynomolgus monkey placenta by immunohistochemistry in the early second trimester.

CITED2 is a conserved regulator of the uterine-placental interface.

Establishment of the hemochorial uterine-placental interface requires exodus of trophoblast cells from the placenta and their transformative actions on the uterus, which represent processes critical for a successful pregnancy, but are poorly understood. We examined the involvement of CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) in rat and human trophoblast cell development. The rat and human exhibit deep hemochorial placentation.

The AKT1-FOXO4 axis reciprocally regulates hemochorial placentation.

Hemochorial placentation involves the differentiation of invasive trophoblast cells, specialized cells that possess the capacity to exit the placenta and invade into the uterus where they restructure the vasculature. Invasive trophoblast cells arise from a well-defined compartment within the placenta, referred to as the junctional zone in rat and the extravillous trophoblast cell column in human. In this study, we investigated roles for AKT1, a serine/threonine kinase, in placental development using a genome-edited/loss-of-function rat model.

Intersection of regulatory pathways controlling hemostasis and hemochorial placentation.

Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI).

ASCL2 reciprocally controls key trophoblast lineage decisions during hemochorial placenta development.

Invasive trophoblast cells are critical to spiral artery remodeling in hemochorial placentation. Insufficient trophoblast cell invasion and vascular remodeling can lead to pregnancy disorders including preeclampsia, preterm birth, and intrauterine growth restriction. Previous studies in mice identified achaete-scute homolog 2 (ASCL2) as essential to extraembryonic development.

Generation of Transgenic Cynomolgus Monkeys Overexpressing the Gene for Amyloid-β Precursor Protein.

Alzheimer's disease (AD) is the most common cause of dementia and understanding its pathogenesis should lead to improved therapeutic and diagnostic methods. Although several groups have developed transgenic mouse models overexpressing the human amyloid-β precursor protein (APP) gene with AD mutations, with and without presenilin mutations, as well as APP gene knock-in mouse models, these animals display amyloid pathology but do not show neurofibrillary tangles or neuronal loss. This presumably is due to differences between the etiology of the aged-related human disease and the mouse models.

Lvrn expression is not critical for mouse placentation.

Preeclampsia is a systemic disease caused by abnormal placentation that affects both mother and fetus. It was reported that Laeverin (LVRN, also known as Aminopeptidase Q) was up-regulated in the placenta of preeclamptic patients. However, physiological and pathological functions of LVRN remained to be unknown.

Trophoblast-Specific Conditional Atg7 Knockout Mice Develop Gestational Hypertension.

Hypertensive disorder of pregnancy (HDP) is a serious pregnancy complication that is life threatening to both the mother and fetus. Understanding HDP pathophysiology is important for developing medical treatments. This study demonstrates the involvement of autophagy deficiency in adverse maternal and fetal outcomes using trophoblast-specific autophagy related (Atg)7, an autophagy-related protein, knockout mice.

Trophoblast-specific Genetic Manipulation Using Lentiviral Delivery.

In this protocol report, we describe a lentiviral gene delivery technique for genetic modification of the rat trophoblast cell lineage. Lentiviral packaged gene constructs can be efficiently and specifically delivered to the trophoblast cell lineage of the blastocyst. The consequences of 'gain-of-function' and 'loss-of-function' blastocyst manipulations can be evaluated with outgrowth assays or following transfer to pseudopregnant rats.

Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction.

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury.

CRISPR/Cas9-mediated mutation revealed cytoplasmic tail is dispensable for IZUMO1 function and male fertility.

IZUMO1 is a protein found in the head of spermatozoa that has been identified as essential for sperm-egg fusion. Its binding partner in the egg has been discovered (JUNO); however, the roles of several domains within IZUMO1 remain unexplored. One such domain is the C-terminus, which undergoes major phosphorylation changes in the cytoplasmic portion of the protein during rat epididymal transit.

CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.

Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals.

Lentiviral Vector-Mediated Complementation Restored Fetal Viability but Not Placental Hyperplasia in Plac1-Deficient Mice.

The X-linked Plac1 gene is maternally expressed in trophoblast cells during placentation, and its disruption causes placental hyperplasia and intrauterine growth restriction. In contrast, Plac1 is also reported to be one of the upregulated genes in the hyperplastic placenta generated by nuclear transfer. However, the effect of overexpressed Plac1 on placental formation and function remained unaddressed.

Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.

Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation.

The effects of chemically synthesized saposin C on glucosylceramide-β-glucosidase.

Objective: Saposin C (SAP-C) is an essential activator of glucosylceramide (GlcCer)-β-glucosidase (GCase), the enzyme deficient in Gaucher's disease. In this study, we investigated the effects of chemically synthesized SAP-Cs (synthetic SAP-Cs) on GCase.

Methods: Enzymatic assays and western blot analyses were carried out to evaluate the effects of two kinds of synthetic SAP-Cs, a non-glycosylated form and a N-glycosylated form bearing a complex type nonasaccharide, on GCase with respect to its activation, stabilization, and protection.

Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes.

The recombinant clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system has opened a new era for mammalian genome editing. Here, we constructed pX330 plasmids expressing humanized Cas9 (hCas9) and single guide RNAs (sgRNAs) against mouse genes and validated them both in vitro and in vivo. When we randomly chose 291 target sequences within protein coding regions of 73 genes, an average number of off-target candidates (exact match 13 nucleotides from 3' target and NGG) found by Bowtie software was 9.